Effects of $Ca^{2+}$ and $HCO_3{^-}$ on Capacitation, Hyperactivation and Protein Tyrosine Phosphorylation in Guinea Pig Spermatozoa

In our previous report, we demonstrated that the tyrosine phosphorylation of sperm proteins (TPSP) of guinea pig was associated with capacitation and hyperactivation (CAHA), and $Ca^{2+}$ and ${HCO_3}^-$ were required for the initiation of CAHA and increasing the TPSP. The aim of this study was to further investigate the mechanism underlying the above events. The results showed that addition of cAMP agonists, dibutyryl-cAMP (db-cAMP) and isobutyl-methylxantine (IBMX), to ${HCO_3}^-$ -free medium significantly increased CAHA to the normal level (when sperm were incubated in TALP). Although addition of the cAMP agonists to $Ca^{2+}$-free medium increased CAHA, the percentages of hyperactivated and capacitated sperm were still significantly lower than the normal level. Compared with ${HCO_3}^-$ -free or $Ca^{2+}$-free medium, TPSP was increased when db-cAMP and IBMX were added in the media. H-89, a specific inhibitor of protein kinase A (PKA), inhibited CAHA in a dose-dependent manner and totally blocked TPSP. These results confirm a previous observation that $Ca^{2+}$ and ${HCO_3}^-$ regulated CAHA and TPSP in a cAMP/PKA pathway, and support an interation between TPSP and CAHA of sperm. Besides the cAMP/PKA pathway, $Ca^{2+}$ might have also played a role in regulating CAHA by other pathways since the normal level of CAHA did not recover by adding cAMP agonists in the media.